Process mining techniques are able to extract knowledge from event logs commonly available in today’s information systems. These techniques provide new means to discover, monitor, and improve processes in a variety of application domains. There are two main drivers for the growing interest in process mining. On the one hand, more and more events are being recorded, thus, providing detailed information about the history of processes. On the other hand, there is a need to improve and support business processes in competitive and rapidly changing environments. This manifesto is created by the IEEE Task Force on Process Mining and aims to promote the topic of process mining. Moreover, by defining a set of guiding principles and listing important challenges, this manifesto hopes to serve as a guide for software developers, scientists, consultants, business managers, and end-users. The goal is to increase the maturity of process mining as a new tool to improve the (re)design, control, and support of operational business processes

Microalgae are regarded as promising organisms to develop innovative concepts based on their photosynthetic capacity that offers more sustainable production than heterotrophic hosts. However, to realize their potential as green cell factories, a major challenge is to make microalgae easier to engineer. A promising approach for rapid and predictable genetic manipulation is to use standardized synthetic biology tools and workflows. To this end we have developed a Modular Cloning toolkit for the green microalga Chlamydomonas reinhardtii. It is based on Golden Gate cloning with standard syntax, and comprises 119 openly distributed genetic parts, most of which have been functionally validated in several strains. It contains promoters, UTRs, terminators, tags, reporters, antibiotic resistance genes, and introns cloned in various positions to allow maximum modularity. The toolkit enables rapid building of engineered cells for both fundamental research and algal biotechnology. This work will make Chlamydomonas the next chassis for sustainable synthetic biology.

Efficient phototrophic production of a high-value sesquiterpenoid from the eukaryotic microalga Chlamydomonas reinhardtii, Metabolic Engineering, http://dx.doi.org/10.1016/j.ymben.2016 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting galley proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. olaf.kruse@uni-bielefeld.de AbstractThe heterologous expression of terpene synthases in microbial hosts has opened numerous possibilities for bioproduction of desirable metabolites. Photosynthetic microbial hosts present a sustainable alternative to traditional fermentative systems, using freely available (sun)light and carbon dioxide as inputs for bio-production. Here, we report the expression of a patchoulol synthase from Pogostemon cablin Benth in the model green microalga Chlamydomonas reinhardtii. The sesquiterpenoid patchoulol was produced from the alga and was used as a marker of sesquiterpenoid production capacity. A novel strategy for gene loading was employed and patchoulol was produced up to 922 ± 242 µg g -1 CDW in six days. We additionally investigated the effect of carbon source on sesquiterpenoid productivity from C. reinhardtii in scale-up batch cultivations. It was determined that up to 1.03 mg L -1 sesquiterpenoid products could be produced in completely photoautotrophic conditions and that the alga exhibited altered sesquiterpenoid production metabolism related to carbon source.

Photosynthetic microbial hosts such as cyanobacteria and eukaryotic microalgae have recently emerged as alternative engineering platforms for the sustainable light-driven bio-production of terpenoids. Many desirable compounds with numerous applications can be produced in microorganisms by heterologous expression of terpene synthases. However, success of green microbial systems has been hampered by issues such as insufficient enzyme expression titers and low flux to desired terpenoid products from carbon fixed during photosynthesis. This work demonstrates how the green microalga Chlamydomonas reinhardtii can be engineered to produce the sesquiterpene biodiesel precursor (E)-α-bisabolene. Through strategic genetic engineering, substantial enhancements of productivity were achieved by coordinated tuning of the isoprenoid metabolism, combining serial enzyme loading for terpene synthase overexpression and amiRNA-based repression of competing pathways. Up to 10.3 ± 0.7mg bisabolene·g cell dry weight could be produced in five days, which represents more than a 15-fold increase over single synthase expression strains. Investigation of strain performance in scale-up cultivations determined overall bisabolene productivity benefits from light:dark cycles. Mixotrophic cultivation can yield up to 11.0 ± 0.5mg bisabolene per liter in seven days in these conditions, and phototrophic production of 3.9 ± 0.2mg per liter was feasible. These achievements represent an important milestone in the engineering of C. reinhardtii towards the goal of designing sustainable, light-driven, green-cell algal bio-factories.

Among green freshwater microalgae, Chlamydomonas reinhardtii has the most comprehensive and developed molecular toolkit, however, advanced genetic and metabolic engineering driven from the nuclear genome is generally hindered by inherently low transgene expression levels. Progressive strain development and synthetic promoters have improved the capacity of transgene expression; however, the responsible regulatory mechanisms are still not fully understood. Here, we elucidate the sequence specific dynamics of native regulatory element insertion into nuclear transgenes. Systematic insertions of the first intron of the ribulose-1,5-bisphosphate carboxylase/oxygenase small subunit 2 (rbcS2i1) throughout codon-optimized coding sequences (CDS) generates optimized algal transgenes which express reliably in C. reinhardtii. The optimal rbcS2i1 insertion site for efficient splicing was systematically determined and improved gene expression rates were shown using a codon-optimized sesquiterpene synthase CDS. Sequential insertions of rbcS2i1 were found to have a step-wise additive effect on all levels of transgene expression, which is likely correlated to a synergy of transcriptional machinery recruitment and mimicking the short average exon lengths natively found in the C. reinhardtii genome. We further demonstrate the value of this optimization with five representative transgene examples and provide guidelines for the design of any desired sequence with this strategy.

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