Wang Jue, Anderson Karl, Yang Ellen, He Lian, Lidstrom Mary E
Department of Chemical Engineering, University of Washington, Seattle, DC, USA.
Synth Biol (Oxf). 2021 Aug 6;6(1):ysab020. doi: 10.1093/synbio/ysab020. eCollection 2021.
Formate is an attractive feedstock for sustainable microbial production of fuels and chemicals, but its potential is limited by the lack of efficient assimilation pathways. The reduction of formate to formaldehyde would allow efficient downstream assimilation, but no efficient enzymes are known for this transformation. To develop a 2-step formate reduction pathway, we screened natural variants of acyl-CoA synthetase (ACS) and acylating aldehyde dehydrogenase (ACDH) for activity on one-carbon substrates and identified active and highly expressed homologs of both enzymes. We then performed directed evolution, increasing ACDH-specific activity by 2.5-fold and ACS lysate activity by 5-fold. To test for the activity of our pathway, we expressed it in a methylotroph which can natively assimilate formaldehyde. Although the enzymes were active in cell extracts, we could not detect formate assimilation into biomass, indicating that further improvement will be required for formatotrophy. Our work provides a foundation for further development of a versatile pathway for formate assimilation.
甲酸盐是可持续微生物生产燃料和化学品的一种有吸引力的原料，但其潜力因缺乏有效的同化途径而受到限制。将甲酸盐还原为甲醛将实现高效的下游同化，但目前还没有已知的高效酶可用于这种转化。为了开发一种两步甲酸盐还原途径，我们筛选了酰基辅酶A合成酶（ACS）和酰化醛脱氢酶（ACDH）的天然变体对一碳底物的活性，并鉴定了这两种酶的活性高且表达量高的同源物。然后我们进行了定向进化，将ACDH的比活性提高了2.5倍，将ACS裂解物活性提高了5倍。为了测试我们途径的活性，我们在一种能够天然同化甲醛的甲基营养菌中表达了该途径。尽管这些酶在细胞提取物中具有活性，但我们无法检测到甲酸盐同化为生物量，这表明甲酸营养还需要进一步改进。我们的工作为进一步开发通用的甲酸盐同化途径奠定了基础。
