Metabolic Engineering of Microorganisms to Produce L-Aspartate and Its Derivatives

Round 1

Reviewer 1 Report

The authors provided comprehensively described the current research progress on microbial production of aspartate and its derivatives in this review article. Additionally, they have discussed the limiting factors of microbial production and provide insight into the industrially relevant strain engineering approaches on how to increase the titer, yield and productivity of the target products, fermentation condition optimization, and downstream purification. I have several minor comments below:

(1) Section 2.2 "Developing cell factories to produce β-alanine":
The reference (https://doi.org/10.1016/j.ymben.2022.08.012) utilized biosensors for screening β-alanine producers. This led to an engineered strain with a high β-alanine yield of 1.08 mol β-alanine/mole glucose producing 6.98 g/L β-alanine in a batch-mode bioreactor, and 34.8 g/L through a whole-cell catalysis using glucose as a sole carbon source. Despite not being the highest titer reported, this is the first work demonstrating the potential of the use of biosensor-enabled high-throughput screening for the production of β-alanine, which provides a blueprint for facilitating strain development. The importance of this concept cannot be neglected. Please add this reference to this section and Table 2.

 

(2)Line 225: Klebsiella pneumoniae is a natural 3-HP producer. However, it is an opportunistic pathogen that can cause different types of healthcare-associated infections, including pneumonia, bloodstream infections, meningitis, and urinary tract infections. Any concern about using this species in the industry, especially for scaling up the production?

 

(3) Line 284-286: The entire sentence "Furthermore, some engineered strain can use glucose as the only substrate....." is redundant. Please provide some insight about why yeast Saccharomyces cerevisiae produces much less D-pantothenic acid than bacteria. Any limited precursor pool in yeast that cannot provide enough flux toward D-pantothenic acid production?

 

(4) Line 317-318: Why is microbial L-homoserine production still challenging in the industry? Have you done a cost comparison between microbial production and chemical synthesis? It would be great to provide some evidence here.

Author Response

The authors provided comprehensively described the current research progress on microbial production of aspartate and its derivatives in this review article. Additionally, they have discussed the limiting factors of microbial production and provide insight into the industrially relevant strain engineering approaches on how to increase the titer, yield and productivity of the target products, fermentation condition optimization, and downstream purification. I have several minor comments below:

(1) Section 2.2 "Developing cell factories to produce β-alanine": 
The reference (https://doi.org/10.1016/j.ymben.2022.08.012) utilized biosensors for screening β-alanine producers. This led to an engineered strain with a high β-alanine yield of 1.08 mol β-alanine/mole glucose producing 6.98 g/L β-alanine in a batch-mode bioreactor, and 34.8 g/L through a whole-cell catalysis using glucose as a sole carbon source. Despite not being the highest titer reported, this is the first work demonstrating the potential of the use of biosensor-enabled high-throughput screening for the production of β-alanine, which provides a blueprint for facilitating strain development. The importance of this concept cannot be neglected. Please add this reference to this section and Table 2.

Thanks. We have added this reference in the section, Table 2 as well as reference.

(2)Line 225: Klebsiella pneumoniae is a natural 3-HP producer. However, it is an opportunistic pathogen that can cause different types of healthcare-associated infections, including pneumonia, bloodstream infections, meningitis, and urinary tract infections. Any concern about using this species in the industry, especially for scaling up the production?

Thanks. Klebsiella pneumoniae is in sure an opportunistic pathogen, and that is one of the reasons that researchers try to develop other cell factories to produce 3-HP. It is not allowed to produce 3-HP with Klebsiella pneumoniae in industry.

(3) Line 284-286: The entire sentence "Furthermore, some engineered strain can use glucose as the only substrate....." is redundant. Please provide some insight about why yeast Saccharomyces cerevisiae produces much less D-pantothenic acid than bacteria. Any limited precursor pool in yeast that cannot provide enough flux toward D-pantothenic acid production?

Thanks. Since kinds of compounds could be converted to D-pantothenic acid by microorganisms, we mentioned “glucose as the only substrate” just for distinguishing them. Compare to E. coli DPA02/pT-ppnk, S.cerevisiae produced less D-pantothenic acid, and there were several reasons: 1several key enzymes were engineered in E. coli DPA02 while they were the wild types in S.cerevisiae; 2in E. coli DPA02, cofactor imbalance problem was solved by overexpression of ppnk (encoding NAD kinase), while in S.cerevisiae, Pos5, the NADH kinase was overexpressed; 3they have used different promoters to Fine-tuning key genes expression. The strength of the promoters may influence the carbon flux involved in D-pantothenic acid biosynthesis; 4these two strains have used different strategies to balance the supply of pyruvate and oxaloacetate. Pyruvate is involved in pantoic acid biosynthesis and oxaloacetate is the substrate for β-alanine biosynthesis. To balance the supply of pantoic acid and β-alanine has great influence on D-pantothenic acid production as they are the two substrates for D-pantothenic acid biosynthesis.

(4) Line 317-318: Why is microbial L-homoserine production still challenging in the industry? Have you done a cost comparison between microbial production and chemical synthesis? It would be great to provide some evidence here.

Thanks. The reported highest productivity is 1.96 g/L/h which is lower than the industry demand (>= 2g/L/h), and the yield is 0.50 g/g which is less than 50% of the theoretical value. Low yield wastes the substrate and also increases the cost, so it is still challenging. I didn’t do a cost comparison between different strategies for L-homoserine synthesis. However, I know it is not produced in large scale.

Reviewer 2 Report

According to the manuscript, metabolic engineering of microorganisms is a potential strategy to produce L-aspartate and its derivatives in a way that is both sustainable economically and environmentally. The authors made an effort to explain the metabolic engineering approaches in L-aspartate (and its derivatives) production in microorganisms with their remaining limitations and possible breakthroughs. However, the manuscript leaves out some crucial information and has a lot of unclear and vague descriptions. Therefore, the manuscript should be thoroughly revised and strengthened while adhering to the pointed guidelines below.

1. Although the manuscript entitled “Metabolic Engineering of Microorganisms to Produce L-aspartate and its Derivatives”, the descriptions related to metabolic engineering strategies are poorly described throughout the manuscript. For each product of interest, the cases are given without sufficient information, such as detailed metabolic engineering strategies used in the study, target genes for the strain engineering and their functionality, culture conditions and scales, and carbon sources (and their proportion if mixed sourced used). Please elucidate the strategies used in the selected references. Moreover, even though it is stated that the current research progress would be presented, the examples, apart from the table, are insufficiently given in the main text. For the comprehension of readers, it is necessary to provide more examples.

2. In the manuscript, the downsides of the conventional production of products are presented without a clear explanation with statistical analysis/data. In addition, the environmental and economic benefits of using bioproduction of the products with proper references are lacking in the manuscript. Thus, please elucidate the factors for the fluidity of contextualization.

3. On page 2, line 80~81, it is described that there is still much work to do for the industrial production of L-aspartate by fermentation because of its low productivity. However, the reason for the “much work to do” is not demonstrated. Likewise, on page 12, lines 317~318, it is stated that there is still a distance to achieve L-homoserine biosynthesis with cell factories in industry, without any detailed explanation. Please include the reasons for the limitations and their possible causes for better contextualization.

4. In the discussion section, some suggested hurdles, including the poor growth of engineered strains and secretion problems of the produced chemicals, are presented without any possible approaches for further improvement. Please include related information on this.

5. On page 13, lines 391~395, it is mentioned that with the development of novel technologies, such as synthetic biology and bioinformatics, the engineering process becomes easier and faster. However, the descriptions/references related to the novel technologies are lacking throughout the manuscript. Please reinforce the examples/explanation of the cutting-edge technologies in synthetic biology and bioinformatics and elucidate the utilization of the progressing techniques in metabolic engineering in the discussion section.

6. Please organize the description of the fermentation strategies in tables uniformly. Also, it would be better to include a column for metabolic engineering strategies used in the references (such as adaptive laboratory evolution, strain modification, gene introduction, etc.) to link the aim of the manuscript.

7. Please correct incorrect spellings (page 1, line 14), non-use of capital letters, and a non-italicized name of the bacterial species (page 6, line 190) presented throughout the manuscript.

none

Author Response

According to the manuscript, metabolic engineering of microorganisms is a potential strategy to produce L-aspartate and its derivatives in a way that is both sustainable economically and environmentally. The authors made an effort to explain the metabolic engineering approaches in L-aspartate (and its derivatives) production in microorganisms with their remaining limitations and possible breakthroughs. However, the manuscript leaves out some crucial information and has a lot of unclear and vague descriptions. Therefore, the manuscript should be thoroughly revised and strengthened while adhering to the pointed guidelines below.

1. Although the manuscript entitled “Metabolic Engineering of Microorganisms to Produce L-aspartate and its Derivatives”, the descriptions related to metabolic engineering strategies are poorly described throughout the manuscript. For each product of interest, the cases are given without sufficient information, such as detailed metabolic engineering strategies used in the study, target genes for the strain engineering and their functionality, culture conditions and scales, and carbon sources (and their proportion if mixed sourced used). Please elucidate the strategies used in the selected references. Moreover, even though it is stated that the current research progress would be presented, the examples, apart from the table, are insufficiently given in the main text. For the comprehension of readers, it is necessary to provide more examples.

Thanks. We added the metabolic strategies for developing each engineered strain in the table, and we also described the genome modifications in detail for the strains with the highest production of the target products in the main text.

2. In the manuscript, the downsides of the conventional production of products are presented without a clear explanation with statistical analysis/data. In addition, the environmental and economic benefits of using bioproduction of the products with proper references are lacking in the manuscript. Thus, please elucidate the factors for the fluidity of contextualization.

Thanks. We have added the description in the text.

3. On page 2, line 80~81, it is described that there is still much work to do for the industrial production of L-aspartate by fermentation because of its low productivity. However, the reason for the “much work to do” is not demonstrated. Likewise, on page 12, lines 317~318, it is stated that there is still a distance to achieve L-homoserine biosynthesis with cell factories in industry, without any detailed explanation. Please include the reasons for the limitations and their possible causes for better contextualization.

Thanks. We have added some descriptions in the manuscript.

4. In the discussion section, some suggested hurdles, including the poor growth of engineered strains and secretion problems of the produced chemicals, are presented without any possible approaches for further improvement. Please include related information on this.

Thanks. In our manuscript, we mentioned that the poor growth could be solved with pathway modification in the section of “Developing cell factories to produce L-aspartate”. For secretion problems, we gave examples that Ghiffary et al. have found a β-alanine exporter in C.glutamicumwhich had a great in-fluence on the titer of β-alanine.

5. On page 13, lines 391~395, it is mentioned that with the development of novel technologies, such as synthetic biology and bioinformatics, the engineering process becomes easier and faster. However, the descriptions/references related to the novel technologies are lacking throughout the manuscript. Please reinforce the examples/explanation of the cutting-edge technologies in synthetic biology and bioinformatics and elucidate the utilization of the progressing techniques in metabolic engineering in the discussion section.

 

Thanks. We gave some examples in the discussion section.

 

6. Please organize the description of the fermentation strategies in tables uniformly. Also, it would be better to include a column for metabolic engineering strategies used in the references (such as adaptive laboratory evolution, strain modification, gene introduction, etc.) to link the aim of the manuscript.

Thanks. We have added one column in each table to describe the metabolic engineering strategies.

7. Please correct incorrect spellings (page 1, line 14), non-use of capital letters, and a non-italicized name of the bacterial species (page 6, line 190) presented throughout the manuscript.

Thanks. We have corrected them.

 

 

Reviewer 3 Report

In this review, the authors gave an insight into the green synthesis of L-aspartate and derivatives regarding developing microbial cell factories using metabolic engineering strategies. It’s a worthwhile work. I have several general comments and suggestions:

 

1. The title should be revised in a more meaningful way. As mentioned in the introduction, metabolic engineering focuses on metabolic pathway modification, while the review summarized cell factories development including not only metabolic-engineered microbial cells but also genetically manipulated cells that could express enzymes for catalysis.

 

2. I think a summary figure that illustrates the biosynthesis of L-aspartate and derivatives that combined Figure 1 and Figure 2 in the introduction first should be much easier to understand.

 

3. The market for chemicals should use data recently updated, but not that years ago, i.e.: Line 53, the global market for L-aspartate was 93.15 million dollars in 2021... Line 105, the global market revenue of β-alanine was 75 million USD in 2019...Line 146, the market for ecotine was 20 million USD 10 2021...Line 259, the global market for D-pantothenic acid was forecast about 460.3 million USD in 2020...

 

4. The table alignment should be improved. Most of them are confusing and parameters cannot be distinguished from one another. Line 82, yield in Table 1 used mol/mol, while in the rest used g/g, no productivity was given as in other Tables. Maleate was used in Table 1 and also the text, while in Figure 1 it was malate, it’s confusing.

 

5. It’s good to group cell factories into metabolic synthesis and whole-cell catalysis as in Table 2, but in other tables, they were not grouped.

 

6. A series of strains corresponding to one platform host were presented in the table, like Table 2, E. coli XBR41, NL-A13, W3110, ALA7, etc. But what are the differences besides titer, yield, and productivity? The authors should explain why they got these different results, and how the cell factories were constructed, by using which approach, instead of repeating data from the table.

 

7. It’s better to change ‘3. Discussion’ into ‘Outlook’ or ‘Perspective’. Although the fermentation condition and downstream processing are important, the review should give more attention to metabolic engineering, like what approaches can be applied for further improvement in the three metrics.

no comments

Author Response

In this review, the authors gave an insight into the green synthesis of L-aspartate and derivatives regarding developing microbial cell factories using metabolic engineering strategies. It’s a worthwhile work. I have several general comments and suggestions: 

1. The title should be revised in a more meaningful way. As mentioned in the introduction, metabolic engineering focuses on metabolic pathway modification, while the review summarized cell factories development including not only metabolic-engineered microbial cells but also genetically manipulated cells that could express enzymes for catalysis.

 Thanks. In this paper, we focused on metabolic engineering of bacteria to produce L-aspartate and its derivatives, and showed the metabolic engineering strategies in all the tables, so we want to keep our title.

2. I think a summary figure that illustrates the biosynthesis of L-aspartate and derivatives that combined Figure 1 and Figure 2 in the introduction first should be much easier to understand.

Thanks. In Fig. 1, we tried to introduce the biosynthetic pathways of L-aspartate with different compounds as substrates. In Fig. 2, we wanted to show the pathways to convert aspartate to its derivatives. Since there were too many pathways involved, we thought it may be easy to follow if we arranged them into two figures.

3. The market for chemicals should use data recently updated, but not that years ago, i.e.: Line 53, the global market for L-aspartate was 93.15 million dollars in 2021... Line 105, the global market revenue of β-alanine was 75 million USD in 2019...Line 146, the market for ecotine was 20 million USD 10 2021...Line 259, the global market for D-pantothenic acid was forecast about 460.3 million USD in 2020...

 Thanks. The information is the up-to-date data we can find on the website.

4. The table alignment should be improved. Most of them are confusing and parameters cannot be distinguished from one another. Line 82, yield in Table 1 used mol/mol, while in the rest used g/g, no productivity was given as in other Tables. Maleate was used in Table 1 and also the text, while in Figure 1 it was malate, it’s confusing. 

 Thanks.

We have modified Table 1 from mol/mol to g/g.

Since the reference didn’t give the productivity information, so we leave them as blank.

Maleate and malate are two different kinds of compounds. We have added Maleate in Fig.1.

5. It’s good to group cell factories into metabolic synthesis and whole-cell catalysis as in Table 2, but in other tables, they were not grouped. 

Thanks. We have modified them according to your suggestions.

6. A series of strains corresponding to one platform host were presented in the table, like Table 2, E. coliXBR41, NL-A13, W3110, ALA7, etc. But what are the differences besides titer, yield, and productivity? The authors should explain why they got these different results, and how the cell factories were constructed, by using which approach, instead of repeating data from the table. 

Thanks. We have added the genetic modifications they have done in different strains in the tables.

7. It’s better to change ‘3. Discussion’ into ‘Outlook’ or ‘Perspective’. Although the fermentation condition and downstream processing are important, the review should give more attention to metabolic engineering, like what approaches can be applied for further improvement in the three metrics.

Thanks. We have changed the topic to ‘Perspective’, and made modifications in the text marked in red.

 

 

Round 2

Reviewer 2 Report

Authors revised their manuscript acceptable for publication.

Reviewer 3 Report

The modifications have been made according to my requirements, and I suggest accepting this article