Synthetic biology approaches for the construction of improved microbial cell factories

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Lopez-Garcia, Carmen Lorena
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Shao, Zengyi
Phillips, Gregory
Gupta, Mohan
Mansell, Thomas
Jarboe, Laura
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Chemical and Biological Engineering
Microbial production of compounds valuable to a myriad of sectors, including chemical, pharmaceutical, nutraceutical, and agricultural industries, has been recognized as a promising strategy for more sustainable sourcing. This approach offers higher flexibility for the synthesis of novel products compared to the traditional chemical route. Furthermore, if researchers aim to succeed at establishing bioproduction at the industrial scale, larger titers of target molecules need to be achieved. Consequently, for organisms to be able to divert their resources towards a desired product, non-trivial manipulation of the cellular metabolism needs to be performed. For a microbial cell factory to be efficient, in addition to the adequate accommodation of the native metabolism for its demanded task, the metabolic flux in a heterologous pathway driving the synthesis of the desired compound needs to be regulated and this is usually accomplished through the variation of familiar genetic parts. However, researchers are aware that the approximately 6000 genes which make every biological function possible in yeast only correspond to the 70% of the genetic information, which opens the door for investigation of the hidden occurrences in the sequence space beyond the characterized genomic parts. In this work, we aim to improve our understanding of noncoding sequences and their contribution in the performance of yeast microbial cell factories. We investigated the impact that context at the sequence level could have on pathway performance. Randomization of the upstream and downstream context of the transcriptional units of the cellobiose pathway in the model yeast Saccharomyces cerevisiae resulted in a more efficient utilization of sugar, indicating an increase in pathway activity which was later verified through transcriptional assays. More intriguingly, further characterization of the sequence context hinted to the potential regulation due to changes in the local organization of DNA, i.e., nucleosome organization. This strategy to diversify expression could be easily applied to other industrially relevant pathways for which screening procedures are in place. Comprehensive understanding of metabolism, abundant tools for genetic manipulation and suitability for industrial processes are a few of the advantages why S. cerevisiae has been traditionally at the front of efforts to construct superior strains. However, it has become clear that the prospect of using less characterized organisms might be worthy, especially when target molecules are less taxing for these organisms to produce due to their inherent metabolic capabilities, compared to model organisms. Yarrowia lipolytica is a nonconventional yeast species that has gained popularity in recent years due to its biotechnological potential and its characteristic feature of lipid accumulation. Further exploration of noncoding sequences as a subject study for this species materialized upon the inspection of a previously disregarded piece of DNA found in the unique structure that autonomous replicating sequences (ARSs) takes in this yeast. Development of stable vectors is a prerequisite for a novel species to be adopted and we found that selection of ARS not only impacts pathway performance but also vector stability. Among the collection of ARSs we evaluated, it was demonstrated that native sequences are more beneficial for bioproduction compared to their synthetic counterparts. Finally, researchers choosing to employ Y. lipolytica as a host should consider the use of the wildtype ARS1 sequence rather than their minimal version ORI1001-CEN1-1 for improved vector maintenance.